Immunotherapeutic compositions for the treatment of alzheimer&#39;s disease

ABSTRACT

A safe and effective vaccine to prevent, slow, halt or reverse progression of Alzheimer&#39;s disease in human patients is disclosed. The vaccine includes Aβ1-42 or an beta amyloid self epitope (e.g. Aβ1-15, or other 7-mer or 15-mer peptide epitopes derived from Aβ1-42) conjugated to an immunogenic carrier (e.g. DT) formulated in a water-in-oil Th2-biased adjuvant/delivery system.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/057,952, filed Feb. 7, 2011, which is a United States NationalStage Application under 35 USC § 371 of PCT/US2009/004504, filed Aug. 6,2009, which claims the benefit of the filing date of U.S. ProvisionalApplication Ser. No. 61,086,938, filed Aug. 7, 2008, the disclosure ofwhich is hereby incorporated by reference.

FIELD OF THE INVENTION

The field relates to a safe and efficacious vaccine to prevent, slow,halt or reverse progression of Alzheimer's disease (AD). AD ischaracterized by the deposition of amyloid β (Aβ) deposits in the brain.Clearance of Aβ by Aβ1-42 vaccination (AN1792) has shown promiseclinically, but suffered from limiting inflammatory effects in a subsetof patients. Self antigens like Aβ are poorly immunogenic, requiringpotent adjuvants such as the QS21, a saponin used in AN1792 vaccine,which induce Th1 biased responses. Alum, an approved Th2 biasedadjuvant, acts poorly with self antigens.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a neurodegenerative disorder characterizedby a progressive loss of cognitive function and is the most frequenttype of dementia in the elderly, affecting almost half of all patientswith dementia.

Correspondingly, advancing age is the primary risk factor for AD. Amongpeople aged 65, 2-3% show signs of the disease, while 25-50% of peopleaged 85 have symptoms of AD and an even greater number have some of thepathological hallmarks of the disease without the characteristicsymptoms. Every five years after the age of 65, the probability ofhaving the disease doubles. The share of AD over the age of 85 is thefastest growing segment of the AD population in the US, although currentestimates suggest the 75-84 year-old population has about the samenumber of patients as the over 85 population (Herbert at al., 2003). TheAlzheimer's Association recently reported that there are more than 5million people in the US living with AD (Alzheimer's Association, 2007).This number is expected to triple by the year 2050.

The current cost to government agencies of the care of patients who haveAD is substantial, and it is rising rapidly: By 2010, Medicare spendingon AD is expected to grow to $49.3 billion (a 54% increase over thecosts in 2000), and Medicaid spending will grow to $33 billion (an 80percent increase over costs in 2000). AD has been reported to costAmerican businesses $61 billion annually. Of that amount, the annualcost attributable to lost productivity and replacement costs whenworkers become caregivers for a relative who has AD is an estimated$36.5 billion. These costs reflect neither the direct financial costs tofamily caregivers (e.g., lost income) nor the costs associated withdepression among family members providing end-of-life care (Prigerson,2003). There is currently no cure for AD and available medications offerrelatively small symptomatic benefit for some patients but do not slowdisease progression.

AD is characterized by the deposition of cerebral amyloid-β (Aβ)protein, neuritic plaques, glial cell activation, and neurofibrillarytangles composed of hyperphosphorylated tau protein (Selkoe, 2001).Epidemiologic, pathologic, and genetic evidence demonstrates that Aβ hasa pivotal role in the pathogenesis of AD (Golde, 2003). Immunization ofamyloid precursor protein (APP) transgenic mice with aggregated Aβ1-42peptide in Freund's adjuvant injected intraperitoneally resulted in thelowering of cerebral Aβ (Schenk et al., 1999). Reduced cerebral Aβlevels in PDAPP-tg mice following intranasal immunization with Aβ1-40peptide have also been reported (Lemere et al., 2000; Weiner et al.,2000). Soon thereafter, several reports demonstrated the importance ofantibody-mediated clearance of Aβ and its role in improving cognition(Bard et al., 2000; Janus et al., 2000; Morgan et al., 2000; Dodart etal., 2002). In addition, anti-Aβ antibodies have been induced followingimmunization with Aβ using various adjuvants (Lemere et al., 2002;Cribbs et al., 2003; Lemere et al., 2000, 2002, 2003; Spooner et al.,2002: Maier et al., 2005; Ghochikyan et al., 2006) and by DNAimmunization (Ghochikyan et al., 2003, Zhang et al., 2003, Okura et al.,2006, Frazer et al, 2007). In addition to an active immunizationstrategy, passive immunization with antibodies against Aβ have also beenshown to remove Aβ from the brain and is associated with an improvementin cognitive function in a mouse model of AD (Bard et al., 2000,DeMattos et al., 2001). Together these encouraging animal data led to amulti-center Aβ vaccine (AN1792) clinical trial (Schenk, 2002; Orgogozoet al., 2003; Gilman et al., 2005).

The AN1792 vaccine was deficient in two respects. First, AN1792 inducedan effective immune response in only 59 of 300 treated patients (19.7%)and, secondly, the clinical trial had to be halted when 18 (6%) of thetreated subjects experienced symptoms of meningoencephalitis (Schenk,2002; Orgogozo et al., 2003; Gilman et al., 2005). Autopsy case reportsfrom subjects who received AN1792 vaccination demonstrated regions withstrongly reduced numbers of plaques compared to controls (Nicoll et al.,2003; Ferrer et al., 2004; Masliah et al., 2005). However, T cellinfiltrates, (primarily CD4⁺ with fewer CD8⁺ cells) were present in theleptomeninges, perivascular spaces, and brain parenchyma in two cases,suggesting a T cell-mediated immune response to AN1792 vaccination.Although no neuroinflammation was observed in pre-clinical studies arecent report had shown that immunization of C57BL/6 mice with Aβ andpertussis toxin induces autoimmune meningoencephalitis withcharacteristics similar to those observed in humans immunized withAN1792 (Furlan et al., 2003). Follow up studies on the AN1792 trialshowed that AD patients that developed antibodies that bound to Aβplaques showed significantly slower rates of decline in cognitivefunction. These findings suggest that the generation of anti-Aβantibodies by active immunization is a promising immunotherapeuticapproach for AD provided that a robust immune response can be elicitedin these elderly patients, and that excessive cell mediated immunereactions are minimized in order to avoid unwanted neuroinflammation.The exact mechanism by which Aβ antibodies reduce Aβ burden in the brainis not known but hypotheses include Fc-receptor mediated phagocytosisvia microglia, dissolution of amyloid fibrils, or sequestration ofcirculating Aβ resulting in an increased net efflux of Aβ from the brain(Vasilevko and Cribbs, 2006). Clearly, whatever the mechanism,immunotherapy, either active or passive, has the potential to clear Aβin AD and improve cognitive function.

Active immunization schedules are being developed to minimize Tlymphocyte-mediated immune reactions and to maximize antibodyproduction. The B cell epitope(s) in humans (Geylis et al., 2005),monkeys (Lemere et al., 2004) and mice (Lemere et al., 2000; McLaurin,et al., 2002; Agadjanyan et al., 2005) is located within the Aβ1-15region, while the T cell epitope has been mapped within Aβ15-42 (Cribbset al., 2003; Monsonego et al., 2003). Thus, Aβ fragments spanning the Bcell epitope but not the T cell epitopes may be safer immunogens topotentially avoid deleterious anti-Aβ cellular immune responses. Manyapproaches to enhance immunogenicity of these shorter Aβ fragments havebeen studied such as conjugation of the B cell epitope, Aβ1-15 to theuniversal helper T cell epitope, PADRE (Agadjanyan et al., 2005;Ghochikyan et al., 2006), expansion with the addition of lysine residuesand glutamate substitutions to reduce β-sheet content (Siguardsson etal., 2001, 2004) or presentation as multiple copies (Zhou et al., 2005).More recently an UBITh® AD immunotherapeutic vaccine has been describedwhereby the intrinsic self Th epitopes of Aβ1-42 are replaced withforeign UBITh® epitopes (Wang et al., 2007). Results from a repeat dosetoxicity study in macaques have shown no evidence for immunotoxicity oroverall toxicity following immunization with this Aβ1-14 UBITh® vaccine.

The dendrimeric Aβ1-15 (dAβ1-15), composed of 16 copies of Aβ1-15 on abranched lysine core which includes an Aβ-specific B cell epitope butlacks the T-cell epitope, is an effective immunogen. Immunizationintranasally (i.n.) with dAβ1-15, using an experimental adjuvant LT(R192G), mutant E. coli heat-labile enterotoxin (Dickinson and Clements,1995), resulted in a robust humoral immune response with a significantreduction in cerebral plaque burden in J20 APP-tg mice (Seabrook et al.,2006). When injected s.c. in mice dAβ1-15, with LT (R192G) adjuvant,induced a humoral immune response with anti-Aβ antibodies, principallyof the IgG1 isotype with lower levels of IgG2a and IgG2b, which boundcerebral Aβ plaques in brain tissue from an AD patient (Seabrook et al.,2006). In another study, the i.n. immunization with a tandem repeat oftwo lysine-linked Aβ1-15 sequences (2× Aβ1-15) using LT (R192G) adjuvantreduced cerebral Aβ load and learning deficits in hAPP mice in theabsence of an Aβ-specific cellular immune response (Maier et al., 2006).

In addition to using short Aβ derivatives that have less intrinsicneurotoxicity, adjuvants which can direct the immune response towards aTh2 phenotype may also be critical for the design of a safe,immunogenically robust, and efficacious vaccine for AD (Cribbs et al.,2003; Ghochikyan et al., 2006). QS21, an adjuvant known to induce astrong Th1 humoral response (IgG2a antibodies in mice) was used in theAN1792 trial, and may have contributed to the T cell mediatedinflammation observed during its clinical evaluation. Many studies inanimals to date focused on getting a good antibody titer by using strongadjuvants, such as CFA/IFA, that give a mixed Th1/Th2 immune response.It has been shown that Aβ1-15 in tandem with the universal helper T cellepitope (PADRE), PADRE Aβ1-15-MAP when formulated in alum, a Th2 biasedadjuvant, generated mainly IgG1 antibody isotypes, but gave a lessrobust immune response than when formulated in Quil A, a Th1 biasedadjuvant generating predominantly IgG2a isotypes (Ghochikyan et al.,2006). Although robust Th-2 type humoral responses to Aβ followingsubcutaneous or intramuscular routes have not been reported, theTh2-type humoral response in APP/TG mice following intranasalvaccination with Aβ peptide using LT (R192G) adjuvant was associatedwith significant decreases in the cerebral Aβ plaque burden, decreasedlocal microglial and astrocytic activation, and reduced neuriticdystrophy (Weiner et al., 2000). Thus, in principle, an anti-Aβ vaccinethat elicits a robust Th2 biased immune response in the AD populationshould be efficacious for treatment of AD.

SUMMARY OF THE INVENTION

Anti-Aβ vaccines that induce a robust Th2 biased immune response whileavoiding deleterious inflammatory Th1 biased cellular immune responsesmay be efficacious for treatment of AD. Aβ1-42 in an appropriateadjuvant may also achieve this result. In addition, B cell-specificshort Aβ peptide epitopes lacking Th1 epitopes are promising candidatesfor an effective immunotherapy regime for AD by avoiding potentiallydeleterious inflammatory cellular immune responses. However, suchpeptide self epitopes require additional T cell help, and additionallyrequire a strong Th2 biased adjuvant to drive the desired immuneresponse and generate high titers of anti-Aβ antibodies in humans. Theinvention provides Aβ peptide epitopes such as Aβ1-42 in a water-in-oilemulsion type Th2-biased adjuvant/delivery system to avoid or suppressTh1 biased cell mediated inflammation. One embodiment of the inventionconcerns the self epitope (Aβ1-15) that lacks the Th1 epitope,conjugated to an immunogenic carrier (DT) to form Aβ15DT conjugate,which is then formulated in a water-in-oil emulsion adjuvant/deliverysystem to induce a Th2 biased immune response. The immunogeniccompositions may be administered to patients by subcutaneous orintramuscular injection in one example.

The disclosed immunotherapeutic compositions relate to immunogenscomprised of Aβ 1-42, or amino acid residues derived therefrom,including amino acid residues 1-15 of Aβ coupled via a C-terminalpeptide spacer to an immunogenic carrier, such as diphtheria toxoid(DT—a component of approved childhood and adult vaccines), formulated ina water-in-oil emulsion type adjuvant. The compositions are designed toinduce neutralizing neutralizing antibodies against AB while avoidingTh1 based cytotoxic T cell mediated inflammation.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B depict anti-Aβ1-42 antibody levels and Ig isotypes inplasma following immunization.

FIG. 2 shows binding of plasma from the mice immunized with Aβ1-42 inCFA/IFA, IFA, or MAS-1 adjuvants to Aβ plaques in sections of brain fromAPP Tg mice.

FIG. 3 shows an immunopotency in mice of Aβ15₍₇₎DT and Aβ15₍₂₂₎DTconjugates at peptide-to-carrier substitution ratios of 7 and 22,respectively, formulated in MAS-1.

FIG. 4 shows an immunopotency in mice of Aβ15₍₇₎DT and Aβ15₍₂₂₎DTconjugates at peptide-to-carrier substitution ratios of 7 and 22,respectively, formulated in MAS-1.

FIG. 5 depicts anti-Aβ isotypes in C57BL/6 mice induced by Aβ1-42,Aβ15₍₇₎DT, and Aβ15₍₂₂₎DT in MAS-1.

FIG. 6 shows an epitope mapping of anti-Aβ specifity induced by Aβ1-42,Aβ15₍₇₎DT, and Aβ15₍₂₂₎DT in MAS-1.

FIG. 7 shows comparable anti-Aβ Immunoreactivity to Human AD plaques byplasma from DBA mice immunized with Anti-Aβ1-42, Aβ15₍₇₎DT, andAβ15₍₂₂₎DT in MAS-1.

FIG. 8 depicts Anti-Aβ levels in 3×Tg-AD mice.

FIG. 9 shows a splenocyte stimulation assay.

FIG. 10 depicts brain sections from each 3×Tg-AD Mouse stained for Aβplaque.

DETAILED DESCRIPTION

The examples and drawings provided in the detailed description aremerely examples, which should not be used to limit the scope of theclaims in any claim construction or interpretation.

AD Therapeutic Vaccines

Conjugation of small molecules, including peptides, to immunogeniccarriers such as DT is an established means of enhancing theirimmunogenicity. However, to render self antigen conjugates stronglyimmunogenic in humans also requires formulation with a suitableadjuvant. MAS-1, is a water-in-oil emulsion adjuvant system comprisingmannide monooleate, squalene and squalane available from Mercia Pharma,Inc. MAS-I was developed for use in humans with self antigen conjugateconstructs to produce therapeutic vaccines that stimulate sustainedneutralizing antibody responses to self antigens without inducingcell-mediated cytotoxicty or breaking immune self tolerance against theself component of the vaccine, and that are well tolerated and withoutsystemic toxicity.

Mannide monooleate based adjuvants are commercially available, such asIncomplete Freund's Adjuvant (IFA) from a number of sources, and ISA 51and ISA 720 available from SEPPIC, Paris, France. Water-in-oil typeemulsion adjuvants may also be formulated with mannide monooleate whichis commercially available from a number of sources (such as Combe, Inc.under the tradename, Arlacel), and squalene and squalane (from severalcommercial sources). The water-in-oil adjuvants used in the disclosedcompositions may be formulated so that the aqueous globules in theemulsion carrying the antigen have median diameters less than 1 micronwith median diameters in the range from about 100 nanometers to about 1micron, and typically with an average diameter of about 300 nanometers.The oil components of MAS-1 are naturally occurring biological oils thatare metabolizable, unlike the mineral oil that comprises the oil phaseof the well known Freund's adjuvants (both incomplete and completeformulations).

A number of carrier proteins such as, but not limited to, diphtheriatoxoid (DT), CRM197 (Wyeth)—a mutant form of DT, tetanus toxoid (TT) andkeyhole limpet hemocyanin (KLH), may be used in the compositions of theinvention. DT is a preferred carrier protein because it is approved foruse in childhood and adult vaccines with an excellent safety record andis produced in quantity to cGMP by a number of commercial sources.

Specificity of the Adjuvant/Delivery System

For decades, Freund's adjuvant emulsions (CFA/IFA) have been standardsagainst which other adjuvants have been measured. CFA is not suitablefor use in humans, due especially to the intense inflammatory reactionsinduced by its mycobacterium component; although, IFA has been used inclinical trials, it has never been approved for any human indications.Nevertheless, these water-in oil (W/O) emulsions are generallyrecognized as potent adjuvants and are used widely in animal studies.

Alum is the only currently U.S.-approved adjuvant with a Th2 biasavoiding cell mediated cytotoxicity, but alum is inadequate as anadjuvant for self-epitope conjugate vaccines due to insufficientimmunopotency. IFA type adjuvants such as ISA 51, which are mineral oilbased, have a drawback as an adjuvant for repeat use in treating chronicdisease settings because IFA's mineral oil deposits remain at injectionsites and can lead to formation of cysts.

MAS-1 adjuvant/delivery system was specifically developed to augmenthumoral responses to poorly immunogenic self-antigens in humans. MAS-1adjuvant emulsions are significantly more potent than Alum andcomparable or superior to IFA emulsions in terms of immunogenicity, butMAS-1 is significantly better tolerated than IFA after i.m or s.c.injection and has excellent pharmaceutical physico-chemicalcharacteristics. These include homogeneous globule size distribution forefficient antigen presentation, low viscosity to facilitate low volumedoses, and extended stability at refrigerated temperatures facilitatingdistribution through standard cold chain procedures.

Unlike IFA, MAS-1 is comprised of natural and metabolizable componentsthat provide a depot of vaccine and thereby promote prolonged efficientimmunostimulation. MAS-1 is eventually cleared from the injection site.MAS-1 emulsions are robust, reproducible and stable whether made in bulkor as single units at point-of-use and may be produced in formulationswith aqueous globules carrying the antigen having median diameters lessthan 1 micron, and typically about 300 nanometers. By contrast, IFAemulsions are administered in formulations that have aqueous globules ofabout 3 to 10, or even 50, microns in diameter with concomitantvariability in emulsion stability, are highly viscous making smallvolume dosing and large scale bulk manufacture difficult.

Aβ1-42 Peptide Vaccine Formulated in MAS-I

The Aβ1-42 vaccine (AN1792) is formulated in a QS21 based adjuvant.QS21, a strong Th1 biased adjuvant, may have contributed to theinflammatory side effects of the AN1792 vaccine. Aβ1-42 formulated inMAS-I is evaluated for its capacity to promote a robust Th2 biasedantibody response to amyloid plaque in brain tissue, while avoiding thegeneration of amyloid beta specific cell mediated immunity associatedwith a Th1 response.

FIGS. 1A and B depict anti-Aβ1-42 antibody levels and Ig isotypes inplasma following immunization:

1 (A) Female DBA mice are immunized s.c. with 100 ug Aβ1-42 on days 0,14, 42 and 84 and anti-Aβ antibodies measured in plasma by ELISA atbaseline, day 28, 56 and 98 and B) Ig isotypes at day 98.

Aβ1-40 and Aβ1-42 peptide antigens are synthesized by standardsolid-phase peptide synthesis methodology. DBA2 mice (n=4) are injectedsubcutaneously (s.c.) with 100 μg Aβ (75 μg Aβ1-40 and 25 μg Aβ1-42)formulated in MAS-1, CFA/IFA, or IFA on days 0, 14, 42 and 84. Antibodytiters in plasma are determined by ELISA on days 0, 28, 56 and 98.Results show that full length Aβ formulated in MAS-1 induces robustantibody titers superior to IFA at day 28, 56 and 98 (FIG. 1A).

Aβ1-40 and Aβ1-42 peptide antigens are synthesized by standardsolid-phase peptide synthesis methodology. DBA2 mice (n=4) are injectedsubcutaneously (s.c.) with 100 μg Aβ (75 μg Aβ1-40 and 25 μg Aβ1-42)formulated in MAS-1, CFA/IFA, or IFA on days 0, 14, 42 and 84. Antibodytiters in plasma are determined by ELISA on days 0, 28, 56 and 98.Results showed that full length AB formulated in MAS-1 induces robustantibody titers superior to IFA at day 28, 56 and 98 (FIG. 1A). Aβ1-42in CFA/IFA (positive control), as expected, yielded a more rapid andinitially higher antibody levels than either the MAS-1 or IFAformulations. The responses to both MAS-1 and IFA formulations increasedthroughout the study and had not reached a plateau at day 98 when thefinal blood sample was taken; Isotyping of the day 98 samples showedthat CFA/IFA elicited a mixed Th1/Th2 immune response with significanttiters of IgG2a and IgG2b antibodies, respectively. Whereas, the MAS-1and IFA formulations elicited Th2 dominated responses, with IgG1 thepredominant isotype along with IgM, low levels of IgG2b, and only verylow levels of the Th-1 type IgG2a antibodies (FIG. 1B). Plasma from themice immunized with full length Aβ1-42 in CFA/IFA, IFA, and MAS-1 showedequal levels of binding to human Aβ plaque in brain sections from APP Tgmice (FIG. 2), demonstrating that the Th-2 dominant antibody isotypeseffectively recognized amyloid plaque indicating that the Th-2 biasedvaccine has the potential to reduce amyloid plaque burden while avoidingTh-1 mediated toxicity.

FIG. 2 shows binding of plasma from the mice immunized with Aβ1-42 inCFA/IFA, IFA, or MAS-1 adjuvants to Aβ plaques in sections of brain fromAPP Tg mice. Plasma are taken at day 98 from mice immunized with Aβ1-42in CFA/IFA, IFA, and MAS-1 on days 0, 14, 42 and 84 equally boundcerebral Aβ plaques in brain sections from APP Tg mice (lower panel).Pre-immune plasma is used as a control and did not bind cerebral Aβplaques (upper panel).

Plasma from the mice immunized with full length Aβ1-42 in CFA/IFA, IFA,and MAS-1 show equal levels of binding to human Aβ plaque in brainsections from APP Tg mice (FIG. 2), demonstrating that the Th-2 dominantantibody isotypes effectively recognized amyloid plaque indicating thatthe Th-2 biased vaccine has the potential to reduce amyloid plaqueburden while avoiding Th-1 mediated toxicity.

AβDT Conjugated Vaccines

Peptide Epitope Selection:

Targeting the Aβ B cell epitope(s) whilst avoiding the Aβ-specific Tcell epitope(s) is a strategy pursued by some investigators to avoidsome of the adverse effects seen in the AN1792 clinical trial withfibrillar, full length Aβ1-42. It has been shown that the Aβ1-15sequence encodes relevant B cell epitopes (Geylis et al., 2005; Lemereet al., 2004; Lemere et al., 2000; McLaurin, et al., 2002; Agadjanyan etal., 2005). This sequence may be conjugated to immunogenic carriers toimprove immunogenicity.

The data presented in FIGS. 1A, 1B and 2 show that when Aβ1-40 andAβ1-42 are formulated in MAS-1 adjuvant or IFA adjuvant, these Aβvaccines may induce Th2 dominated immune responses. Whereas, Aβ1-40 andAβ1-42 formulated in CFA also induces a significant Th1 immune responsewhich results in Th1 cell mediated inflammation as reported with Aβ1-40and Aβ1-42 formulated in QS21 adjuvant in AN 1792 vaccine. Thus, basedon these results, conjugated Aβ vaccines comprising Aβ epitope sequencesderived from Aβ amino acid residues 16 through 40 and 16 through 42,when conjugated to a suitable immunogenic carrier may be expected toinduce robust and safe Th2 dominated immune responses when formulated asMAS-1 or IFA based vaccines. Likewise, these constructs when formulatedwith alum, an approved Th2 biased adjuvant, may also be expected toproduce Th2 dominated immune responses. These epitope sequences maytypically contain from 7 to 15 consecutive amino acid residues derivedfrom the Aβ sequences 1-40 and 1-42.

Immunogenic Carrier Selection:

Aβ1-15 peptide lacking helper T cell epitopes is poorly immunogenic whenformulated in Th-2 biased alum adjuvant (Agadjanyan et al., 2005). Thisis likely to be the case for Aβ1-15 peptide formulated in MAS-1, sinceAβ1-14 formulated in IFA has been shown to be a poor immunogen in guineapigs unless extrinsic T cell help was provided by coupling to keyholelimpet hemocyanin (KLH) carrier protein or to foreign UBITh epitopes(Wang et al., 2007). Similarly, Aβ peptides comprising 7 to 15 aminoacid residues derived from Aβ1-40 and Aβ1-42 are also predicted to bepoorly immunogenic when formulated by themselves in IFA, MAS-1, or alumadjuvants.

The immunogenicity of short non-immunogenic peptides may generally beenhanced by coupling to Th epitopes such as synthetic PADRE constructs(Agadjanyan et al., 2005), or to immunogenic carrier proteins, e.g.mutant cholera B toxin (CBT), KLH, mutant diphtheria toxin (CRM), or totoxoids such as tetanus toxoid (TT) or diphtheria toxoid (DT), all ofwhich contain Th epitopes to provide T cell help for IgG production andimmunological memory. DT is chosen as the immunogenic carrier in this ADvaccine, because it has long been approved for use in childhood andadult vaccines and is available as a GMP compliant component. In oneembodiment, the Aβ peptide epitope is conjugated to DT via a sevenresidue spacer sequence with a terminal cysteine residue via itssulfhydryl moiety using a bi-functional cross-linker. In otherembodiments, the Aβ epitopes without the 7 residue spacer sequence butending in a terminal cysteine residue may be conjugated via itssulfhydryl moiety to the immunogenic carrier. Alternative couplingchemistries well known in the art, such as carbodiimide chemistries, mayalso be used to effect the conjugation of Aβ epitopes to the immunogeniccarrier.

Synthesis of Aβ15DT Conjugates:

The results indicate that immunogenicity may be affected by thepeptide-to-carrier substitution ratio. The Aβ15 peptide sequence and theconjugation methods, are provided below. In summary, a 22 residueAβ15-mer peptide is synthesized by solid-phase chemistry. Aβ15DTconjugates are prepared at two Aβ15 peptide:DT molar substitutionratios. The substitution ratios of the two Aβ15DT conjugates, determinedby mobility on SDS-PAGE, are 7.6 (#1) and 21.1 (#2) (see Table 1).Conjugation ratios from about 5 moles to about 30 moles of peptide permole of immunogenic carrier are useful for the compositions.

TABLE 1 Characterization of Aβ15DT Conjugates by SDS-PAGE DT Aβ15DT #1Aβ15DT #1 Median MW 55.1 kD 75.6 kD 111.8 kD Molar Sub. Ratio(peptide:DT) NA 7.6 21.1

Immunopotency of Aβ15DT in MAS-1:

Both young DBA2 (6 wk-old; n=4/group) and aged C57BL/6 (12 mo-old;n=2/group) female mice are immunized with the conjugates at 100 μg dosesin 0.1 mL MAS-1, s.c., on days 0, 14, 42, and 84. Blood samples aretaken pre-immunization and at days 28, 56, and 98, and Aβ antibodytiters are assayed by ELISA using Aβ1-42 peptide as target antigen.Results are presented in FIG. 4.

FIG. 4 shows an immunopotency in mice of Aβ15₍₇₎DT and Aβ15₍₂₂₎DTconjugates at peptide-to-carrier substitution ratios of 7 and 22,respectively, formulated in MAS-1. Aβ15DT conjugates are synthesized atpeptide to DT substitution ratios of 7 and 22 moles/mole. The conjugatesand Aβ1-42 are formulated in MAS-1 adjuvant and evaluated at 100 μg/0.1mL s.c dose, injected on days 0, 14, 42 and 84, for immunopotency in 6wk old DBA (n=4/group) and 12 mo old C57BL/6 (n=2/group) mice. Plasma Aβantibodies are measured by ELISA against Aβ1-40.

Both Aβ15₍₇₎DT and Aβ15₍₂₂₎DT conjugates in MAS-1 induced rapid andpotent antibody responses to Aβ measured by ELISA. Anti-Aβ titerscontinued to rise following further immunizations with Aβ15₍₂₂₎DT inMAS-1. The induction of anti-Aβ specific antibodies is significantlysuperior to that seen with Aβ1-42 in MAS-1, which in DBA strain mice areroughly 50 μg/mL on day 28 as shown in FIG. 1. At 28 days, both Aβ15DTconjugates generate anti-Aβ antibody levels more than 5 times greaterthan those generated with Aβ1-42 in MAS-1 in DBA mice. These results,confirm that Aβ15DT in MAS-1 is a highly effective immunogen, even inolder C57BL/6 mice which are in general poorly immunoresponsive to Aβpeptides and, surprisingly, show that the potency of the immune responseincreased as the molar substitution ratio of epitope to immunogeniccarrier was increased.

The results indicate that immunogenicity may be affected by thepeptide-to-carrier substitution ratio, the dose, and the dose regimen.Both conjugates in MAS-1 induced predominantly Th-2 antibody isotypes(FIG. 5) that recognize the N terminal region of Aβ1-42 (FIG. 6), andbind to amyloid plaques in paraffin-embedded human AD brain tissuesections (FIG. 7).

FIG. 6 shows an epitope mapping of anti-Aβ specifity induced by Aβ1-42,Aβ15₍₇₎DT, and Aβ15₍₂₂₎DT in MAS-1. Epitope mapping conducted byinhibition ELISA using Aβ1-40 coating Ag wells and Aβ peptide fragmentsas inhibitors of mouse Ab binding. Similar Ab isotype and specificityresults are obtained with DBA mice.

FIG. 3 shows an immunopotency in mice of Aβ15₍₇₎DT and Aβ15₍₂₂₎DTconjugates at peptide-to-carrier substitution ratios of 7 and 22,respectively, formulated in MAS-1. DA (6 wk-old; n=4/group) and agedC57BL/6 mouse (12 mo-old; n=2/group) female mice received 100 μg of eachconjugate in 0.1 mL MAS-1 s.c. on days O and 14. Blood samples are takenpre-immunization and at Day 28 and Aβ antibody titres assayed by ELISA.

A 0.4:1 w/w mixture of Aβ15₍₇₎DT and Aβ15₍₂₂₎DT in MAS-1 elicitedsignificant anti-Aβ Ab responses in 14 month old 3×Tg-AD mice (FIG. 8).These animals are immunized s.c with 4 doses of 100 ug Aβ15DT in 0.1 mLMAS-1 at 0, 2, 6, and 12 weeks and are euthanized at 16 weeks (i.e., at18 months age). Splenocytes from immunized animals specificallyresponded to Aβ15DT, but not to full length Aβ1-40/42 demonstrating thatimmune tolerance to native Aβ was preserved (FIG. 9). Brain sections(6/mouse) from each animal from the Aβ15DT treated and MAS-1 placebogroups revealed a 74% reduction (p 0.0543 one-tailed Student's t Test)in amyloid plaque burden by Aβ15DT in MAS-1 compared with MAS-1 placebo.At 14 months age in 3×Tg-AD mice amyloid plaque deposition in thehippocampus is well established. Virtual absence of significant amyloidplaque in the vaccinated group demonstrates that immunization withAβ15DT in MAS-1 resulted in a reduction in amyloid plaque and did notsimply prevent further build up of amyloid plaque, indicating thepotential utility for Aβ15DT/MAS-1 immunization to both prevent andtreat Alzheimer's disease.

FIG. 8 depicts anti-Aβ levels in 3×Tg-AD mice. Two age matched groups of3×Tg-AD mice (14 mo old; n=4/group; 3 M, 1 F) are immunized s.c. with100 μg/0.1 mL comprised of a 0.4:1 w/w mixture of the Aβ15₍₇₎DT andAβ15₍₂₂₎DT conjugates in MAS-1 or MAS-1 placebo at 0, 2, 6, 12, and 16wk. Animals are euthanized after 17 weeks immunotherapy (Active18.5 andplacebo 18.3 mo. age respectively). Anti-Aβ1-40 antibodies aredetermined by ELISA on blood samples collected at 0 (pre-immunization),4, 8, and 16 wks.

These animals are immunized s.c with 4 doses of 100 ug Aβ15DT in 0.1 mLMAS-1 at 0, 2, 6, and 12 weeks and are euthanized at 16 weeks (i.e., at18 months age). Splenocytes from immunized animals specificallyresponded to Aβ15DT, but not to full length Aβ1-40/42 demonstrating thatimmune tolerance to native Aβ are preserved (FIG. 9).

FIG. 9 shows a splenocyte stimulation assay. Two age matched groups of3×Tg-AD mice (14 mo old; n=4/group; 3 M, 1 F) are immunized s.c. with100 μg/0.1 mL comprised of a 0.4:1 w/w mixture of the Aβ15₍₇₎DT andAβ15₍₂₂₎DT conjugates in MAS-1 or MAS-1 placebo at 0, 2, 6, 12, and 16wk. Animals are euthanized after 17 weeks immunotherapy (Active18.5 andplacebo 18.3 mo. age respectively).

Brain sections (6/mouse) from each animal from the Aβ15DT treated andMAS-1 placebo groups revealed a 74% reduction (p 0.0543 one-tailedStudent's t Test) in amyloid plaque burden by Aβ15DT in MAS-1 comparedwith MAS-1 placebo. At 14 months age in 3×Tg-AD mice amyloid plaquedeposition in the hippocampus is well established. Virtual absence ofsignificant amyloid plaque in the vaccinated group demonstrates thatimmunization with Aβ15DT in MAS-1 results in a reduction in amyloidplaque and did not simply prevent further build up of amyloid plaque,indicating the potential utility for Aβ15DT/MAS-1 immunization to bothprevent and treat Alzheimer's disease.

Immunogenicity and efficacy may be affected by any number of factorssuch as the peptide-to-carrier substitution ratio, the adjuvant, theformulation of the water-in-oil emulsion, the dose, and the doseregimen. This indicates that these factors must be assessed in order tooptimise AD vaccine efficacy.

FIG. 10 depicts brain sections from each 3×Tg-AD Mouse stained for Aβplaque shows significant reduction in hippocampal plaque burden inanimals immunized with Aβ15DT conjugates in MAS-1, but not in animalsimmunized with MAS-1 placebo.

Our results indicate that immunogenicity and efficacy may be affected byany number of factors such as the peptide-to-carrier substitution ratio,the adjuvant, the formulation of the water-in-oil emulsion, the dose,and the dose regimen. This indicates that these factors must be assessedin order to optimise AD vaccine efficacy.

The Water-in-Oil Emulsion Adjuvant/Delivery System

Many factors, such as antigen, adjuvant, and delivery systems may bemodified to elicit specific cellular and humoral immune responses. Thedata show that the Aβ1-42 formulated in a water-in-oil adjuvant deliverysystem such as MAS-1 induces a significant humoral antibody response innaive mice with a predominantly Th2 bias (IgG1 and lgG2b isotypes)(FIGS. 1 and 2).

However, short Aβ fragments, while potentially avoiding an Aβ-specificcellular immune response, are poorly immunogenic. Conjugation of smallmolecules, including peptides, to immunogenic carriers such as DT is anestablished means of enhancing immunogenicity, but even DT conjugatedself epitopes may require a Th-2 biased adjuvant with superior potencythan alum adjuvant in order to be effective therapeutically. Awater-in-oil adjuvant emulsion such as MAS-1 may induce robust Th2biased immune responses to Aβ15DT conjugated self antigens while havingthe potential to avoid Th1 biased cell mediated inflammatory sideeffects that have limited the effectiveness of previous attempts todevelop and Aβ15DT vaccine for Alzheimer's disease.

In one example, the components of the oil adjuvant vehicle suitable foruse in the compositions, comprise a first sugar ester emulsifier such asmannide monooleate (MMO) or sorbitan monooleate, a second emulsifiersuch as a hydrogenated castor oil, for example, polyoxyl-40-hydrogenatedcastor oil (POCO), and naturally occurring and metabolizable oils,preferably squalene and squalane. The metabolizable oils typicallycomprise from about 85% to about 90% by weight of the oil, the firstsugar ester emulsifier from about 9% to about 12% by weight of the oil,and the second emulsifier from about 0.5% to about 0.7% by weight of theoil. In one example, the metabolizable oil component is typically 50%squalene, 50% squalane by weight, but the concentration of thesecomponents may vary within this component. A suitable adjuvant vehiclefor use in the compositions is MAS-1, which is comprised of naturallyoccurring and metabolizable components derived from vegetable sources,and is commercially available from Mercia Pharma, Inc, Scarsdale, N.Y.(www.merciapharma.com).

The components of the oil vehicle, including their starting materials,may be derived from either animal or vegetable sources, or combinationsthereof, are all commercially available from multiple sources. Suitablesugar esters as the first emulsifier in addition to MMO includepolysorbates, particularly sorbitan monooleate. In addition to POCO asthe second emulsifier, sorbitan esters, such as sorbitan monopalmitate,polysorbates, such as the Tweens family of emulsifiers, and HypermersB239 and B246 may be useful.

In one example, the nanoparticulate vaccine emulsions disclosedtypically contain from about 65% by weight to about 75% by weight of theadjuvant oil vehicle and from about 25% to about 35% by weight of anaqueous phase containing the protein antigen. In certain embodiments ofthe compositions, the aqueous phase comprises from about 27% to about33% by weight of the vaccine emulsion.

The water-in-oil vaccine emulsions used in the compositions, in oneexample, may be formulated so that the aqueous globules in the emulsioncarrying the antigen have median diameters less than 1 micron withmedian diameters in the range from about 100 nanometers to about 1micron, and typically with an average diameter of about 300 nanometers.In one example, the oil components of the adjuvant are preferablynaturally occurring biological oils that are metabolizable, unlike themineral oil that comprises the oil phase of the well known Freund'sadjuvants (both incomplete and complete formulations).

The disclosed vaccine emulsions may tolerate high concentrations ofantigen (up to at least 10 mg/mL) and should be compatible with commonlyused protein solubilizers (e.g., 4M urea, 30% DMSO). Unlike IFAemulsions, in one example, they should be compatible with aqueous phaseshaving a wide range of pH (3-8), and be unaffected over a wide rangesalt concentrations. Unlike IFA emulsions (>1,500 cP), the vaccineemulsions in one example, should have a low viscosity (<100 cP) as freeflowing emulsions permitting high precision low volume (0.1 mL) dosing.The physico-chemical characteristics of the disclosed vaccine emulsions,in one example, should have a median distribution of globule sizediameter of (D(v,0.5)) less than or equal to 1.0 μm, and be unaffectedby high concentrations of protein in the aqueous phase.

Animal Models for Evaluating the Immunogenic Compositions

Gene-targeted and transgenic mice are a valuable tool for modelingvarious aspects of AD pathology, although no mouse model fullyreproduces its entire neuropathology. The 3×Tg-AD mice develop bothplaques composed of Aβ peptide, and neurofibrillary tangle composed ofhyperphosphorylated Tau protein in relevant brain regions, withassociated age-dependent decline in the cognitive phenotype in bothspatial and contextual learning and memory paradigms. Thus, they providea valuable model for evaluating potential AD therapeutics (Oddo et al.,2003).

The peptide immunogen Aβ1-2 has been shown to elicit therapeutic anti-Aβantibodies in preclinical and clinical studies and Aβ1-42 in MAS-1induces Th2 biased anti-Aβ antibodies that recognize amyloid plaques inbrain tissue slices.

3×Tg-AD triple transgenic mice, express human mutant APP, tau andpresenilin 1. These mice originated on a C57BL/6/1295 background buthave been backcrossed for many generations onto C57BL/6 mice, resultingin very little 129 genotype remaining. They are homozygous, easy tobreed and progressively develop AB and tau pathology with a temporal andregional specific profile that closely mimics pathological developmentin the human AD brain. Aβ deposition develops in these mice before thetau pathology, which is consistent with the amyloid cascade hypothesis,which stipulates that Aβ is the trigger and that tau pathology is adownstream consequence of Aβ pathology (Hardy and Selkoe, 2002).

In order to further evaluate the disclosed immunotherapeuticcompositions, one may immunize 3×Tg-AD mice at 6 months(young/prevention) and 14 months of age (old/treatment) by s.c.injections with the optimal dose determined form dose ranging doseregimen studies with Aβ1-15:DT optimized for peptide to carriersubstitution ratio. The mice may be immunized for nine months in thepreventative study and for 5-6 months in the treatment study. A group ofmice vaccinated with Aβ1-42 in MAS-1 could be included as positivecontrol and a negative control group immunized with DT in the selectedadjuvant or W/O placebo adjuvant, in both prevention and treatmentstudies. Due to the variability in the behavioral component of thestudy, group sizes of 16 3×Tg-AD mice will be required.

Behavioral testing using the Morris Water Maze, in one example, shouldbe conducted at the end of each study. Spatial learning may be measuredby latency and distance to platform, while memory retention may bemeasured by probe trials, although correlations between behavioralperformance and amyloid plaque deposition are well established in3×Tg-AD mice.

Immunization of 3×Tg-AD mice with Aβ15DT in MAS-1 lead to a robust Th-2dominated humoral immune response, avoiding Th-1 dominated immunity andthe potential for inducing cell-mediated inflammatory changes in thebrain. The induction of Aβ specific regulatory T cells by thecompositions of the invention should further reduce the potential forcell mediated inflammatory side effects. The antibodies generated leadto a decrease of Aβ in the brain which should correlate with improvementin cognitive function in mice. It was expected that immunizing miceafter they have accumulated cerebral Aβ (14 months) would only partiallylower plaque burden, since it is well recognized that older 3×Tg-AD micetypically express a less robust immune response whereas, in fact, potentTh-2 dominant humoral immune responses and concomitantly statisticallysignificant reductions in hippocampal plaque burden without evidence formicrohemorrhage, are seen.

In the three autopsy cases from the AN1792 clinical trial, extensivevascular Aβ remained despite parenchymal Aβ clearance, and one of thesehad numerous brain microhemorrhages (Nicoll et al., 2003; Ferrer et al.,2004; Maliash et al., 2005). In evaluating the immunotherapeuticcompositions one should play close attention to the appearance ofcerebral microhemorrhage as this has also been observed followingpassive immunization of mice with Aβ monoclonal antibodies (Pfeifer etal., 2002, Wilcock et al., 2004, Racke et al., 2005; Lee et al., 2005).These microhemorrhages are believed to be caused by the overly rapidclearance of Aβ parenchymal deposits and their subsequent vasculardeposition by the high doses of high affinity mAbs, as well as possiblyby the binding of Aβ mAbs with Aβ in the microvasculature.

In general, active immunization in mice has not been associated with thedevelopment of cerebral microhemorrhage, except for a recent reportshowing that active immunization of APP+PS1 transgenic mice with Aβ1-42formulated in CFA/IFA was associated with an increase inmicrohemorrhages (Wilcock et al., 2007). A recent study by Asuni et al.,(2006) showed that vaccination of Tg2576 'mice with an Aβ derivative inalum, an adjuvant favoring a Th2 response, reduced Aβ burden without anyevidence for microhemorrhages. Thus, unlike in the case of Aβ1-42 inQS21 based adjuvant (AN1792), or other Th-1 biased adjuvant formulationsused to promote robust immune responses to Aβ, the results observedherein with Aβ1-42 in MAS-1 or Aβ15DT in MAS-1 in 3×Tg-AD mice, predictthat these compositions are suitable as therapeutic vaccines for earlyprevention and treatment of Alzheimer's disease and are expected toinduce significantly more robust, Th-2 dominated immune responsesavoiding the potential for Th-1 mediated cytotoxicity or breaking immuneself tolerance to the endogenous target while reducing or preventingamyloid plaque deposition and having the potential to reduce theconsequent development of hyperphosphorylated Tau protein.

Materials and Methods

Aβ1-42 Peptide:

The 42 residue peptide may be manufactured using solid phase synthesis.The sequence of this peptide is shown below:

-   -   DAEFR⁵HDSGY¹⁰EVHHQ¹⁵KLVFF²⁰AEDVG²⁵SNKGA³⁰IIGLM³⁵VGGVV⁴⁰IA⁴² (SEQ        ID NO: 1)

Aβ Peptide Epitopes:

The 22 residue 15-mer Aβ peptide immunomimic peptides with a 7 aminoacid residue peptide spacer (XXXXXXC-COOH) may be manufactured usingsolid phase synthesis. The sequences of the 15-mer Aβ1-15, Aβ16-30,

-   Aβ21-35, and Aβ31-42 are shown below:

(SEQ ID NO: 2) NH2-D¹AEFR⁵HDSGY¹⁰EVHHQ¹⁵X: XXXXXC-COOH (SEQ ID NO: 3)NH2-K¹⁶LVFF²⁰AEDVG²⁵SNKGA³⁰ XXXXXXC-COOH (SEQ ID NO: 4)NH2-A²¹EDVG²⁵SNKGA³⁰IIGLM³⁵ XXXXXXC-COOH (SEQ ID NO: 5)NH2-I³¹IGLM³⁵VGGVV⁴⁰IA⁴²XXXXXXC-COOH

Various spacer peptides are known to those skilled in the art and theseand other peptide sequences may be used in the invention. U.S. Pat. Nos.5,023,077, 5,468,494, and 5,688,506 and the disclosures of which arehereby incorporated by reference, describe useful peptide spacersequences that may be used in the disclosed compositions. Wherein thepeptide spacer sequences incorporated from the aforementioned patentsare as follow:

(SEQ ID NO: 6) Arg-Pro-Pro-Pro-Pro-Cys-; (SEQ ID NO:7)Ser-Ser-Pro-Pro-Pro-Pro-Cys-; (SEQ ID NO: 8)Cys-Pro-Pro-Pro-Pro-Ser-Ser-; (SEQ ID NO: 9)Arg-Cys-Pro-Pro-Pro-Pro-Arg-;

AβDT Conjugates:

Methods of conjugating peptides to immunogenic carrier proteins are wellknown to those skilled in the art, for example U.S. Pat. Nos. 5,468,494,5,688,506 and 6,359,116 the disclosure of which are hereby incorporatedby reference.

Aβ1-15 Peptide Synthesis and Aβ15DT Conjugation:

The 22 mer peptide comprising Aβ residues 1-15 may be coupled to DTusing maleimide-NHS ester bifunctional cross-linking chemistry. Thepeptide via its C-terminal CySH residue may be coupled to DT carrier bybifunctional cross-linking agents well known to those skilled in theart, for example, epsilon-maleimidocaproic acid N-hydroxysuccinimideester (eMCS) crosslinker, and related bifunctional analogs (Sulfo-eMCS).This mechanism of coupling has been selected because it is highlyspecific. The DT is first activated with eMCS under pH conditionsenabling reaction of the succinimidyl moiety of the linker to free aminogroups on DT to produce maleimido-activated DT (MDT). Once complete,unreacted eMCS and its degradation products are removed and activationbuffer exchanged for coupling buffer optimal for reaction of the freesulfhydryl of the peptide with the maleimido group of MDT to conjugatethe peptide to the carrier. The conjugate is then purified and analyzed.Selection of the proper eMCS: DT ratio and activation/couplingconditions result in consistent peptide: carrier substitution ratios atboth laboratory and production batch scales. Each conjugate may becharacterized by analytical methods. The peptide: carrier molarsubstitution ratio may be determined by quantitative amino acid analysisand/or by mobility on SDS-PAGE.

Conjugate purity may be assessed by SEC HPLC and by SDS PAGE. Conjugateidentity may be tested by Western Blot utilizing additional samples ofgel from the SDS PAGE and by amino acid analysis.

Aβ1-15BSA Conjugate:

Aβ1-15 epitope, without the spacer sequence but with a C-terminal Cysresidue, is conjugated to BSA, for use as the target antigen in ELISAfor the measurement of peptide specific antibodies. Alternatively,Aβ1-15 peptide, or longer peptides of Aβ starting from the N terminusand including residues up through residue 42, may serve as targetantigens in the ELISA.

Animal Immunizations:

Mice are injected subcutaneously (s.c.) into the scruff of the neck orhind limb flanks, or intraperitoneally (i.p.), with 100 μl of immunogen,by well known methodology.

Blood and Tissue Collection:

Mice are bled from the tail vein prior to and during the immunizationperiod, and serum prepared and frozen at −20° C. For collection oflarger volumes of serum, mice are terminally bled by cardiac puncture atthe end of the experiment and serum prepared as above and used as areference standard for experiments. 3×Tg-AD mice are euthanized by C0₂inhalation and pericardially perfused with saline. The brains areremoved and divided in half along the midline. One hemibrain issnap-frozen in liquid nitrogen and stored at −80° C. for biochemicalstudies (e.g., Aβ ELISAs). The other hemibrain is either drop-fixed in4% paraformaldehyde in PBS for 2 hr at RT, sucrose protected in 10-30%sucrose at 4° C. and embedded in OCT (TissueTek) for cryosectioning or,drop-fixed in 10% neutral buffered formalin for 2 hr at RT, washed inTBS, dehydrated, cleared of lipids (Histoclear), and embedded inparaffin for paraffin sagittal sectioning. Six-to-ten micron sagittalcryosections and 12-micron paraffin serial sections are cut and storedfor staining. Spleen tissue for proliferation and cytokine analyses areremoved under sterile conditions.

Mouse Splenocyte Proliferation Assay:

Splenocytes are prepared by centrifugation of a single cell suspensionon Lympholyte-M (Cederlane, Homby, ON, Mayada). Splenocytes, at aconcentration of 2×10⁶/ml, are cultured in RPMI supplemented with 10%FBS and stimulated with Aβ1-15, Aβ1-42, and DT and Aβ15DT (0-50 μg/ml)in 96 well plates. Conmayavalin A is used as a positive control toensure the viability of the cells. Culture supernatants are harvestedfor cytokine ELISAs at 48 hr. After 72 hr in culture, 1 μCi [3H]thymidine is added to each well and the cells cultured for an additional18 hr. The cells are harvested using a plate washer and theradioactivity measured using a liquid scintillation counter. Astimulation index (SI) is calculated using the following formula: countsper minute (CPM) with peptide antigen/CPM with no antigen.

Measurement of anti AB antibodies in Mouse Sera by ELISA:

Plates are coated with normal mouse IgG (standard curve) and 2 μg/mlAβ1-42 peptide and incubated overnight at 4° C. Plates are then blockedin 5% goat serum, 1% BSA, and 0.005% Tween-20 for 2 hours at RT.Following washing, dilutions of mouse sera is added to the wells andincubated for 2 hours at RT. Goat anti-mouse conjugated to HRP(Kirkegaard and Perry Laboratory, Gaithersburg, Md.) is used assecondary antibody and incubated on plates for 1 hour at RT. Followingaddition of the color substrate, 3,3′,5.5′-Tetramethylbenzidine (TMB)for 30 min the reaction is stopped with 0.5M HCl and the plates read ona plate reader at 450 nm. Isotype Analysis: Quantitative isotypespecific ELISAs are performed using isotype specific secondaryantibodies for IgG1, IgG2a, IgG2b, IgA and IgM (Zymed, San Francisco,Calif.) and the addition of a standard curve of the appropriate isotype(Southern Biotechnology, AL) to the standard ELISA described above.

Detection of Aβ Plaques in Human and Mouse Brain:

Sera from immunized and control mice are diluted serially 1:100 to1:10,000 and applied to human AD and J20APP transgenic mouse brainsections for immunohistochemical detection of plaques and vascularamyloid. Biotinlyated goat anti-mouse secondary antibody is usedtogether with the ABC ELITE HRP standard (A and B) and reacted with DABfor visualization. Adjacent sections are labeled with AB antibodies suchas R1282 (generic AB polyclonal, Selkoe lab) or 6E10 (monoclonal Aβ1-17,Covance Research Products, Dedham, Mass.).

Aβ Protein ELISA: Both soluble and insoluble Aβ40 and Aβ42 levels in thebrain are determined by ELISA kits (Signet) according to themanufacturer's instructions (Covance Research Products, Inc, Berkeley,Calif.). Snap frozen whole hemispheres are homogenized in 4 volumes ofPBS containing protease inhibitor cocktail (Roche, Indianapolis, Ind.).Homogenates are spun at 100×g for 30 min at 4° C. Supernatants areanalyzed for soluble Aβ levels by ELISA. The PBS·pellet are re-suspendedin 10 volumes of guanidine buffer (5M guanidine HCL, 50 mM Tris pH 8.0).Samples are mixed for 4 hr at RT. Brain homogenates are then diluted1:10 in casein buffer (0.25% casein, 5 mM EDTA, protease inhibitorcocktail in PBS), mixed, and spun at 16,000×g for 20 minutes. Additionaldilutions are made in 0.5 M guanidine buffer with 0.1% BSA and insolubleAβ levels measured by ELISA

Immunohistochemistry, Histology, and Image Analysis:

Immunohistochemistry are performed using the ELITE ABC method of VectorLaboratories (Burlingame, Calif.) and DAB as the chromagen. Briefly,paraffin sections are deparaffinized in Histoclear (NationalDiagnostics, Atlanta, Ga.) and rehydrated in graded ethanols to water.Cryosections are thawed, air-dried for 15 min, and washed gently in TBS.From this point on, the staining procedure are the same for bothparaffin sections and cryosections. Endogenous methanol is quenched,sections are blocked in 10% serum in TBS, and sections incubated inprimary antibody overnight at 4° C.

Sections are then washed in TBS, incubated in biotinylated secondaryantibody (Vector Labs) for 30 min at RT, washed in TBS, incubated inavidin-HRP complex (Vector Labs) for 30 min at RT and then developed inDAB.

Sections are counterstained in hematoxylin (unless designated for imageanalysis), dehydrated, cleared and coverslipped with Permount (Fisher).Double immunofluorescence are performed by blocking in 2% serum, mixing2 primary antibodies (MoAb and PAb) and applying to sections overnightat 4° C., rinsing in 0.1 M Tris, blocking in 2% serum in Tris, mixingtwo fluorescently-labeled secondary antibodies and applying to sectionsfor 2 hr at RT, rinsing twice in Tris, incubating the sections in 0.3%Sudan Black B in 70% ethanol in the dark for 10 min, washing in TBS,washing in water, fixing in formalin for 1 hr in the dark, washing inwater, and coverslipping the slides with Hydromount non-fluorescingaqueous media (National Diagnostics, Atlanta).

Coverslips are sealed with clear nail-polish to prevent drying. Negativecontrols for IHC and IF included omission of primary antibody or usingmouse IgG as primary antibody. The primary antibodies forimmunohistological detection of Aβ and markers of inflammation arelisted below.

Primary Antibodies for Immunohistochemistry:

Aβ (R1282, Dr. Selkoe, Brigham and Women's Hospital, Boston); Aβ40, Aβ42and 6E10 (BioSource and Covance Research Products), Glial fibrillaryacidic protein (GFAP) Mab (Dako, Denmark), microglia/macrophage Mab[CD45 (Serotec), CD11b/Mac-1 (Serotec), MHC II (PharMingen), F4/80(Serotec), FcgR II-CD16 and II-CD32 (PharMingen)], B cells (CD40,Novacastra Laboratories, UK), T cells (CD5, CD3; Serotec), APP Mab (IG6,Covance Research Products), phospho-tau (ATB, Innogenetics), mouse 1g(goat anti-mouse IgG).

Histological Staining:

Thioflavine S staining to detect fibrillar Aβ protein in brain sectionsare performed by incubating slides in a 1% aqueous solution ofThioflavine S for 10 min followed by rinses in 80 and 95% ethanol, andthen distilled water. Haemosiderin staining to detect microhemorrhagesare performed by incubating hydrated sections in 2% ferrocyanide in 2%hydrochloric acid for 15 min. Hematoxylin and eosin (H&E) staining areused to assess mononuclear cell infiltration in brain, lung, heart,liver and spleen sections.

Quantitative Image Analysis:

Computer-assisted quantification of Aβ plaque burden and gliosis areperformed using IP Lab Spectrum 7.1 Image Analyzer (Fairfax, Va.) aspreviously described (Weiner et al., 2000). Four-to-eight sagittalimages of immunolabelled sections are captured at equi-distant levels(˜100 μm apart) through each mouse brain using a Nikon microscope with aLeica motorized stage and a SPOT camera. All images for one experimentare captured on the same day with the threshold being held constantthroughout the image analysis. The percent area occupied byimmunoreactivity (above threshold) is calculated.

Alternative combinations and variations of the examples provided willbecome apparent based on this disclosure. It is not possible to providespecific examples for all of the many possible combinations andvariations of the embodiments described, but such combinations andvariations may be claims that eventually issue.

The invention claimed is:
 1. An immunotherapeutic composition for thetreatment of Alzheimer's disease comprising: an immunogen comprising apeptide comprising an amino acid sequence of 7-15 consecutive amino acidresidues of human amyloid beta 1-15 peptide (SEQ ID NO: 2) conjugated toan immunogenic carrier protein, formulated in a water-in-oil emulsionwith an oily adjuvant vehicle comprising squalene, squalane and mannidemonooleate, and polyoxyl-40-hydrogenated castor oil, wherein theimmunogen is contained within aqueous globules that have a mediandiameter from about 100 nanometers to about 1 micron; wherein: theimmunogenic carrier protein is diphtheria toxoid carrier; the immunogenhas a conjugation ratio of moles of peptide per mole of carrier of fromabout 5:1 to about 30:1; the oily adjuvant vehicle comprises from about85% to about 90% squalene and squalene, from about 9% to about 12%mannide monooleate and from about 0.5% to about 0.7% polyoxyl40-hydrogenated castor oil; and the peptide comprising the amino acidsequence of human amyloid beta protein is coupled to the immunogeniccarrier protein via a spacer.
 2. The composition of claim 1 wherein theaverage aqueous globule diameter is about 300 nanometers.
 3. Thecomposition of claim 1, wherein the water-in-oil emulsion comprises fromabout 65% to about 75% oily globules containing the immunogen.
 4. Thecomposition of claim 1, wherein the squalene and squalene are in a 50/50ratio by weight.
 5. The composition of claim 4, wherein the oilyadjuvant vehicle is Mercia Adjuvant System (MAS-1).
 6. The compositionof claim 1, wherein the spacer is a peptide spacer.
 7. Theimmunotherapeutic composition of claim 1, wherein the immunogen has aconjugation ratio of moles of peptide per mole of carrier of 7:1.
 8. Theimmunotherapeutic composition of claim 1, wherein the immunogen has aconjugation ratio of moles of peptide per mole of carrier of 22:1. 9.The immunotherapeutic composition of claim 1, wherein the immunogen hasa conjugation ratio of moles of peptide per mole of carrier of 22:1within the range of about 5:1 to about 25:1.
 10. The immunotherapeuticcomposition of claim 1, comprising a plurality of immunogens accordingto claim 1, said immunogens having conjugation ratios of moles ofpeptide per mole of carrier within the range of about 5:1 to about 25:1.11. A method of treating or reducing amyloid plaque deposition inAlzheimer's disease in a human patient, which comprises administering tothe patient the immunotherapeutic composition of claim 1.